Semiautomated fluorometric analysis of nucleic acids in tissue homogenates

Abstract

This report describes a technique that was developed to provide an efficient and accurate estimation of RNA:DNA ratios. These ratios have been used as an instantaneous measure of recent growth of individual aquatic organisms where morphometrics are not appropriate (e.g., field‐collected species) or insufficiently sensitive (e.g., small life stages or species). In this semiautomated, sensitive method, ethidium bromide fluorescence was used to quantitate total nucleic acids in crude homogenates. Individual concentrations of RNA and DNA were determined by differences in fluorescence before and after elimination of RNA by digestion with RNase. Efficiency of the procedure was enhanced using a computer‐driven multiwell plate scanning system (CYTOFLUOR, Millipore Corporation ) to measure fluorescence at timed intervals and perform data manipulations. Routinely, detection limits of 0.1 μg DNA and 0.4 μg RNA were achieved, allowing the analysis of small, individual organisms. Fluorescence results of split samples were comparable with those obtained using a standard spectro‐photometric method to quantitate nucleic acids. Coefficients of variation for replicate samples within an assay (1.6%) and for samples within replicate assays (5.6%) indicated good test reproducibility. Quantitative recoveries of nucleic acid standards spiked into tissue homogenates were generally high, averaging 91.0% for DNA and 119.0% for RNA. Factors affecting the fluorescence of ethidium bromide stained nucleic acids—e.g., nucleic acid source, crude homogenate components, and buffer composition—are discussed relative to assay performance. This method provides a rapid and reliable assessment of individual growth, an important sublethal toxicological end point, that is suitable for both laboratory and field studies. © 1994 by John Wiley & Sons, Inc..

RNA:DNA ratios as indicators of nutritional condition in the copepod Calanus finmarchicus

“…In order to measure RNA and DNA of very small individual copepods, we adapted the fluorometric assay of Bentle et al (1981) to a 96-well microplate format. The resulting technique, the microplate fluorescent assay (MFA), is similar to that of Nacci et al (1994), who first used a microplatebased RNA-DNA assay with marine animals. Our MFA is capable of measuring nucleic acids in individual copepodite stages and late naupliar stages of C. finmarchicus.…”

Effects of preservation and storage of microcrustaceans in RNAlateron RNA and DNA degradation

“…Both nucleic acids are involved in the protein synthesis and cellular multiplication required for growth, and therefore, the ratio of RNA to DNA has been regarded as an index of protein synthetic capacity per cell (Bergeron 1997;Buckley et al 1999). RNA and DNA can be assayed on whole tissue homogenates (Bentle et al 1981), and over the last several years methods have been developed to enhance precision and accuracy of nucleic acid quantification in small-bodied aquatic organisms, e.g., fluorometric assays in combination with sensitive fluorochromes, use of microplates, and more efficient extraction procedures (Caldarone and Buckley 1991;Nacci et al 1994;Wagner et al 1998;Caldarone et al 2001;Gorokhova and Kyle 2002). These methods provide aquatic ecologists with powerful tools to quantify nucleic acids in as little sample material as a single Daphnia egg (Gorokhova and Kyle 2002) that can be used to investigate the links between environmental factors and plankton growth, physiological requirements, and, ultimately, community structure and functioning (Elser et al 2000).…”